PROJECT DESCRIPTION The trophectoderm (TE) is an extraembryonic tissue that supplies instructive signals required for embryo patterning during gastrulation and gives rise to the placenta, an organ that connects the developing fetus to the uterine wall to allow nutrient uptake, waste elimination, gas exchange and thermoregulation via the mother's blood supply. In mice, trophoblast stem cells (mTSCs) derived from the preimplantation blastocyst or from fibroblasts via direct lineage conversion have become an important tool with which to dissect the mechanisms responsible for TE specification, differentiation and function. However, attempts to establish TSCs with features of early human TE have been unsuccessful. Our proposal seeks to fill this gap in knowledge by establishing induced human TSCs (i-hTSCs) through the process of lineage conversion. To better understand how TSCs form in the human embryo we analyzed previously collected single cell RNA sequencing data from human blastocyst stage embryos. We identified a set of transcription factors that are highly expressed in the TE but not in the embryonic epiblast (EPI) cells. We also identified several signaling ligands expressed in the EPI and/or in the TE itself that may be responsible for specification and/or maintenance of TSCs. To facilitate lineage conversion experiments we have generated human embryonic stem cells (hESCs) that carry a fluorescent mCherry reporter linked to the endogenous GATA2 or GATA3 genes which are highly active in all cells of the TE. We have differentiated these cells to obtain ESC-derived human fibroblast-like cells. Transduction of these reporter cells with the cocktail of doxycycline (Dox)-inducible TE-specific transcription factors yielded several GATA2/3-mCherry positive clones. These clones could be passaged in the presence of Dox and expressed the key genes known to regulate TSC self-renewal. Based on these data, we hypothesize that hTSCs can be induced from fibroblasts and /or hESCs via direct lineage conversion with a cocktail of TE- specific factors in the presence of maintenance cytokines and/or signaling inhibitors. This hypothesis will be tested via two specific aims. In Aim 1, we will develop protocols for direct lineage conversion into i-hTSCs from existing hESCs and/or fibroblasts: (1A) Define the minimal set of conversion factors required for induction of hTSCs from hESCs or fibroblasts; and (1B) Define a set of cytokines and/or signaling inhibitors which allow Dox-independent maintenance of i-hTSCs. In Aim 2, we will perform functional and molecular characterization of i-hTSC lines. Our approach is innovative, because it will develop new biological reagents that would allow derivation and maintenance of early i-hTSCs. The proposed research is significant, because it would enable better understanding of fundamental biology of blastocyst lineages in humans. Such knowledge has the potential to inform new treatments of pregnancy complications, infertility and personalized medicine approaches in reproductive biology.